This proposal addresses autocrine/paracrine actions of catecholestrogens in the porcine ovarian follicle. Our data have shown that these novel compounds have substantial potential as local ovarian regulators since they are synthesized in the ovarian follicle and act to promote differentiation of ovarian follicular cells. In the projected period of support, we will aim at a more detailed and integrated understanding of this putative regulatory system at each of several points which may determine their impact. The biosynthesis of catecholestrogens will be further studied by understanding the nature and regulation of the key biosynthetic enzyme -- estrogen-2-hydroxylase (E-2-H). To approach this issue, the hormonal regulation of this enzyme will be studied in cultured granulosa and theca cells and in follicular tissue from gonadotropin-treated swing. Levels of the catecholestrogens themselves will also be determined in follicular flui and culture medium. The nature of the ovarian E-2-H and its mRNA will be studied with antibodies and cDNA probes. Once validated, such reagents wil lead to detailed studies of enzyme biosynthesis and its regulation. Next, we will study the significance of the metabolism of catecholestrogens in th ovary, since degradation of these steroids is important to their levels and action. For these studies, we will examine CE metabolism via catechol-O- methyltransferase (COMT), its location and hormonal control. Further, we will determine the mechanism of action of 2-O-methyl-estradiol, the chief product of COMT. Finally, we will determine the cellular locus and mechanism of action of the catecholestrogens on ovarian granulosa cells. In these studies, we will evaluate the effects of catecholestrogens on the activity and biosynthesis of the enzymes of the progestin biosynthetic pathway. In particular, effects of catecholestrogens on cyclic AMP generation, and in regulation of HMG-CoA-reductase and cholesterol side- chain cleavage will be determined. These studies are expected to establish a unique role for these compounds in the development of the preovulatory follicle.